Abstract
Recombinant protein expression for structural and therapeutic applications requires the use of systems with high expression yields. Escherichia coli is considered the workhorse for this purpose, given its fast growth rate and feasible manipulation. However, bacterial inclusion body formation remains a challenge for further protein purification. We analyzed and optimized the expression conditions for three different proteins: an anti-MICA scFv, MICA, and p19 subunit of IL-23. We used a response surface methodology based on a three-level Box-Behnken design, which included three factors: post-induction temperature, post-induction time and IPTG concentration. Comparing this information with soluble protein data in a principal component analysis revealed that insoluble and soluble proteins have different optimal conditions for post-induction temperature, post-induction time, IPTG concentration and in amino acid sequence features. Finally, we optimized the refolding conditions of the least expressed protein, anti-MICA scFv, using a fast dilution protocol with different additives, obtaining soluble and active scFv for binding assays. These results allowed us to obtain higher yields of proteins expressed in inclusion bodies. Further studies using the system proposed in this study may lead to the identification of optimal environmental factors for a given protein sequence, favoring the acceleration of bioprocess development and structural studies.
Highlights
Recombinant protein expression for structural and therapeutic applications requires the use of systems with high expression yields
This led us to develop a single chain variable antibody, isolated from a phage display library, directed against the recognition interface between MHC class I chain–related protein A (MICA) and NKG2D; by preventing MICA-mediated NKG2D downregulation, this scFv could potentially serve as therapy in MICA expressing cancers9. scFvs are composed of variable regions from heavy and light chains from immunoglobulins, and fused with a flexible linker
Results anti-MICA scFv, MICA and IL-23p19 are expressed as inclusion bodies in E. coli
Summary
Recombinant protein expression for structural and therapeutic applications requires the use of systems with high expression yields. We optimized the refolding conditions of the least expressed protein, anti-MICA scFv, using a fast dilution protocol with different additives, obtaining soluble and active scFv for binding assays These results allowed us to obtain higher yields of proteins expressed in inclusion bodies. Attractively simple, this is a limited methodology, given the complex nature of the determinants of protein expression, www.nature.com/scientificreports solubility and folding In this scenario, OFAT is not the most efficient approach to obtain information on the operation space, as changing one input can have unexpected effects on the outcomes of other, unrelated, variables[6]. This protein format can be expressed in E. coli, and direct modification of its amino acidic sequence can be carried out for affinity maturation[10]
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